Korean J. Chem. Eng., 34(12), 3156-3162 (2017) pISSN: 0256-1115 DOI: 10.1007/s11814-017-0241-8 eISSN: 1975-7220 INVITED REVIEW PAPER INVITED REVIEW PAPER Anti-melanogenic effect of Prunus davidiana extract in melan-a melanocyte through regulation of OCA-2, TRP-1 and tyrosinase Birendra Kumar Singh, Vivek Kumar Morya†,‡, Hyang-Bok Lee, Jun-Shub Kim, and Eun-Ki Kim† Department of Biological Engineering, Inha University, Nam-gu, Incheon 402751, Korea (Received 21 September 2016 • accepted 5 September 2017) Abstract−Prunus spp. and locally available plants (used as folkloric medicine) were screened to find a novel and nat- ural anti-melanogenic agent. Based on p-protein promoter reporter assay (PPRA) the candidate plants were screened in the quest for p-protein inhibitor. Expression profiling of key proteins revealed the molecular mechanism of the mela- nin inhibition as well as TEM analysis revealed melanosome structure. The screened plant extract through PPRA showed significant down regulation of p-protein, which led to melanin inhibition. Another key melanosomal protein like tyrosinase and TRP-1 was also found to be down-regulated. However, TRP-2 was not affected. TEM analysis of treated cells also revealed that the stage IV melanosomes were lowered in number compared to control. The present study shows the plants used in this study possess good anti-melanogenic properties. However, the P. dav idi an a has the highest anti-melanogenic property among screened plant extracts. Keywords: Pink-eyed Dilution (p) Protein, Melanosome, TEM, P. d av i di an a , Anti-melanogenic Agent INTRODUCTION study, the role of p-protein in melanogenesis is still not clear. How- ever, several reports have been presented on the role of p-protein Melanin is a good devil, which is produced to combat against in melanogenesis: sorting of tyrosinase to melanosomes [6], mem- radiation-induced skin damage. However, overproduction causes brane transport [7] stabilization of melanosomal protein complex skin tanning and results in a psychopathic complexity and other [5], regulation of melanosomal pH [8], arsenic sensitivity [5] and pathological symptoms, such as post inflammatory melanoderma, control of tyrosinase processing and cellular glutathione metabo- melasma, freckles, and solar lentigo. However, cosmetics provide an lism [5,9] and may involve in processing and trafficking of pro- alternative, especially in hiding the phenotype but not a solution. teins to the melanosome. It has been hypothesized that the OCA2 Some fairness cosmetics which inhibit melanin synthesis have chemi- gene encodes a transport or pore protein vital for melanosome cal constituents and mainly inhibit tyrosinase. Tyrosinase is a key function [6,7,10-12]. Based on previous studies [2], it was aimed enzyme in melanogenesis; however, this enzyme also plays a vital to screen locally available various folkloric medicinal plants in a role in other physiological activities. Thus, a long-term use of such quest of p-protein inhibitor. cosmetics causes various side effects, so a melanosome-specific tar- In this study, three plant-extracts from Rosaceae family were get based natural anti-melanogenic agent is being required [1,2]. used; this family includes 95 to 100 genera with several species of Among various proteins associated in melanogenesis a very few economic importance, particularly edible fruit and ornamentals. are melanosomal proteins, and targeting these proteins may pro- These plants are also used for medicinal and nutraceutical prop- vide more safe and effective cosmetics. However, these melanoso- erty, especially in Chinese and Korean-Hanbang traditional medi- mal proteins are also shared by other cells; therefore, it is important cines [13]. Among the screened plants, the P. d av i di an a (Rosaceae) to target a melanosome-specific protein to develop a targeted cos- has received more attention in Korea and is widely cultivated as an metic. Among various known melanosomal proteins the p-pro- ornamental plant, especially in Korea, Japan, Taiwan and China. tein (product of OCA-2) could be a safe target to design a new The stem of P. d av i di an a has been used as folkloric medicine to cosmetic. In previous study in our lab [2,3] we successfully demon- treat for neuritis, antidiabetic, anti-inflammatory and rheumatism strated that p-protein can be a better target. P-protein is an 833- in Korea [14,15]. No specific hazardous effect has been reported amino acid long membrane protein, which consists of a 12 mem- for this species; however, most members of the genus Prunus pro- brane spanning domain [4]. Mutation or deletion in this gene duce hydrogen cyanide. It is usually present in too small a quan- causes oculocutaneous albinism II (OCA2), one of the most com- tity to do any harm; in small quantities, hydrogen cyanide has been mon types of albinism [5]. Due to a lack of direct evidence based shown to stimulate respiration and improve digestion, and is also claimed to be of benefit in the treatment of cancer. At a very high †To whom correspondence should be addressed. concentration, it can cause respiratory failure and even death; how- E-mail: [email protected], [email protected] ever, this is very rare from any natural source (http://www.pfaf.org/ ‡Present Address: DST-Centre for Policy Research, BBAU, Luc- USER/Plant.aspx?LatinName=Prunus+davidiana). know, 226025, India Our aim was to screen selected plants based on the literature Copyright by The Korean Institute of Chemical Engineers. (use as traditional application and local availability), and further to 3156 Anti-melanogenic effect of Prunus davidiana extract in melan-a melanocyte through regulation of OCA-2, TRP-1 and tyrosinase 3157 characterize the mechanism of action. Because, the work is a part (RMPI) was added. After 24 h, 1.0 µg/mL of puromycin was added of comprehensive screening of folkloric medicinal plants in a quest to the medium for selection of transformed cell. Transfected cells of p-protein inhibitors. Therefore, a luciferase based promoter- were subcultured when cells were 70-80% confluent. After four reporter screening assay was performed and the respective cyto- weeks of starting selection, the cell which was grown was ana- toxicity toward Melan-a cells was also evaluated. To understand the lyzed for promoter assay by using the luciferase reporter gene. mechanism of action of the melanin inhibition, the RT-PCR for 3. Measurement of the Luciferase Activity and Cell Viability OCA2 expression, Western blot for MITF, TRP-1, TRP-2 and Assay Tyrosinase was performed. To ensure the cellular physiology and Melan-a cell transfected with pEZX-PG02 was seeded in 96 melanosome structural integrity, TEM analysis was performed to well plates at a density of 1.5×104 cells. At 24 h after seeding, the elucidate the role of plant extracts in melanogenic pathways. cells were treated with each sample for 48 h. The media in which cells grew were collected to measure luciferase activity, and cells on MATERIALS AND METHODS the bottom of plates were used to analyze cell viability. Luciferase activity was measured by using Secrete PairTM Gaussia Luciferase 1. Materials and Reagents Assay Kit (GeneCopoeia, USA). Luicferase activity was measured Roswell Park Memorial Institute-1640 (RPMI-1640), fetal bovine as relative light units (RLUs) emitted when 10 µL media were mixed serum (FBS), trypsin EDTA (TE), phosphate buffered saline (PBS), with 100 µL coelenterazine solution. The luminescence was meas- Penicillin-Streptomycin, Lipofectamine LTX&PLUS reagent, PVDF ured by a luminometer (TD-20e, TURNER BIOSYSTEMS, USA). membrane were purchased from Invitrogen Corp. (CA, U.S.A.). Cell viability was assayed by using MTT reagent. The medium Sodium bicarbonate, 4-(2-hydroxyethyl)-1-piperazineethanesul- was changed with 100 µL of 0.5 mg/mL MTT solution and the cells fonic acid (HEPES), L-DOPA (3, 4-dihydroxy-1-phenylalanine), were incubated for 3 h. MTT solution was removed and 200 µL of dimethyl sulfoxide (DMSO), (3-(4,5-dimethylthiazol-2-yl)-2,5-di- DMSO was added to dissolve the formazan produced in the cells. phenyltetrazolium bromide (MTT), protein cell extraction kit, The absorbance of each well was then read at 540 nm [16]. Phenylmethanesulfonylflouride (PMSF) were obtained from Sigma 4. Cell Viability Assay Chemical Co. (St. Louis, USA). Goat polyclonal NCKX5, Tyrosni- The MTT based assay was used to determine the cell viability nase, Actin antibodies as well as rabbit-antigoat and donkey anti- of melan-a cells. Melan-a cell was seeded in 96 well systems at the goat were purchased from Santa Cruz Biotechnology Inc.,; Quai- concentration of 0.5×104 cells/well respectively, for 24 h and then ® gen RNA extraction kit, Quaigen RT-PCR kit, QuantiTect SYBR treated with the plant extract (0-100 µg/mL) in RPMI-1640 for Green PCR Kit were purchased from, Quaigen. Primers were pur- 48 h. Then again the cells were treated and placed in 10% CO2 for chased from Bioneer (Daejeon, Korea). A luciferase assay kit 24 h. Then medium was replaced with RPMI-1640 with 0.5 µg/ TM µ (Secrete Pair Gaussia Luciferase Assay Kit) was purchased from mL (100 L) MTT and kept in the 10%CO2 incubator for 2-3 h. GeneCopoeia (USA). Further, media containing MTT were removed and 200 µL of 2. Cell Culture and Plant Extract 100% DMSO was added to the cells and cell viability was assessed Melan-a immortal murine melanocytes were purchased from by absorbance at 540 nM using an ELISA reader (Spectra Max ATCC (American Type Culture Collection) and cultured in RPMI- 340 microplate reader) [17]. Untreated cells were used as a con- 1640 medium supplemented with 2g/L of Sodium Bicarbonate, trol group. 6g/L of HEPES, 10% (v/v) FBS, and 200nM TPA at 37oC in a 5.